Abstract
For ease of detection, soluble forms of phage-displayed scFv antibodies are usually expressed with a tag, e.g., c-myc or His (Histidine). The binding is then assayed by a monoclonal antibody to the tag. In this article, we describe the use of biotinylated antigen for detecting soluble scFv antibodies without utilizing the peptide tag detection system. The scFv antibodies were against the oncoplacental antigen heat-stable alkaline phosphatase (HSAP). The method essentially consisted of either reverse Western or antigen capture enzyme-linked immunosorbent assay (ELISA). In the reverse Western, periplasmic extract was electrophoresed, and binding to biotinylated antigen was detected by the detection system based on streptavidin-horseradish peroxidase. The antigen capture ELISA utilized the binding of periplasmic extract to a polystyrene plate. We have also demonstrated the use of antigen capture ELISA for studying specificity and affinity of the selected clones. Although these techniques have been developed for antibodies to HSAP, they have general utility for phage expression systems without a peptide tag.
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Nissim, A., Hoogenboom, H. R., Tomlinson, I. M., Flynn, G., Midgley, C., Lane, D., and Winter, G. (1994), EMBO J. 13, 692–698.
Harrison, J. L., Williams, S. C., Winter, G., and Nissim, A. (1996), Methods Enzymol. 267, 83–109.
Lindner, P., Bauer, K., Krebber, A., Nieba, L., Kremmer, E., Krebber, C., Honegger, A., Klinger, B., Mocikat, R., and Pluckthun, A. (1997), Biotechniques 22, 140–149.
Mack, M., Riethmuller, G., and Kufer, P. (1995), Proc. Natl. Acad. Sci. USA 92, 7021–7025.
Lee, W. S., Kao, C. C., Bryant, G. O., Liu, X., and Berk, A. J. (1991), Cell 67, 365–376.
Bischoff, K. M., Shi, L., and Kennelly, P. J. (1998), Anal. Biochem. 260, 1–17.
Epenetos, A. A., Munro, A. J., Tucker, D. F., Gregory, W., Duncan, W., MacDougall, R. H., Fause, M., Travers, P., and Bodmer, W. F. (1985), Br. J. Cancer 51, 641–644.
Fishman, W. H. (1990), Clin. Biochem. 23, 99–104.
Hirano, K., Domar, U. M., Yamamoto, H., Brehmer Andersson, E. E., Wahren, B. E., and Stigbrand, T. I. (1987), Cancer Res. 47, 2543–2546.
Koshida, K. and Wahren, B. (1990), Urol. Res. 18, 87–92.
Nouwen, E. J., Buyssens, N., and DeBroe, M. E. (1990), Cell Tissue Res. 260, 321–335.
Vergote, I., Onsrud, M., and Nustad, K. (1987), Obstet. Gynecol. 69, 228–232.
Kala, M., Bajaj, K., and Sinha, S. (1997), Anal. Biochem. 254, 263–266.
Marin, M., Brockly, F., Noel, D., Julan, M. E., Piechaczyk, M. B., Hua, D. T., Gu, Z. J., and Piechaczyk, M. (1995), Hybridoma 14, 443–451.
Laemmli, U. K. (1970), Nature (London) 227, 680–685.
Marks, J. D. (1995), in Antibody Engineering, Borrebaeck, C. A. K., ed., Oxford University Press, New York, pp. 53–88.
Muller, R. (1980), J. Immunol. Methods 34, 345–352.
Kazemier, B., de Haard, H., Boender, P., van Gemen, B., and Hoogenboom, H. (1996), J. Immunol. Methods 194, 201–209.
Goldberg, M. E. and Djavadi-Ohaniance, L. (1993), Curr. Opin. Immunol. 5, 278–281.
Van Heyningen, V., Brock, D. J. H., and Van Heyningen, S. (1983), J. Immunol. Methods 62, 147–153.
Friguet, B., Chaffotte, A. F., Djavadi-Ohaniance, L., and Goldberg, M. E. (1985), J. Immunol. Methods 77, 305–319.
Hardy, F., Djavadi-Ohaniance, L., and Goldberg, M. (1997), J. Immunol. Methods 200, 155–159.
Akerstrom, B., Nilson, B. H., Hoogenboom, H. R., and Bjorck, L. (1994), J. Immunol. Methods 177, 151–163.
Loomans, E. E., Roelen, A. J., Van Damme, H. S., Bloemeers, H. P., Gribnau, T. C., and Schielen, W. J. (1995), J. Immunol. Methods 184, 207–217.
Nieba, L., Krebber, A., and Pluckthun, A. (1996), Anal. Biochem. 234, 155–165.
Schuck, P. (1996), Biophys. J. 70, 1230–1249.
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Kala, M., Bajaj, K. & Sinha, S. Direct antigen capture by soluble scFv antibodies. Appl Biochem Biotechnol 90, 11–22 (2001). https://doi.org/10.1385/ABAB:90:1:11
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DOI: https://doi.org/10.1385/ABAB:90:1:11